Prostate cancer is the third leading cause of cancer death in American men because of the eventual failure of androgen deprivation therapy. Mechanisms underlying progression to the hormone refractory stage have not been completely elucidated, but current evidence suggests that activation of the androgen receptor (AR) in the low androgen environment may play a critical role in this process. Our goal is to understand the regulation of AR signaling by tyrosine kinases. Our studies focused on two kinases, HER-2 and Ack1 (activated cdc42-associated kinase). Activation of HER-2 by the ligand heregulin increases AR transactivation and cell proliferation and leads to recruitment of AR to the androgen responsive enhancer. Inhibition of HER-2 impairs AR transcriptional function by decreasing recruitment of AR to the androgen responsive enhancer. New evidence indicates that activated Ack1 promotes androgen-independent progression of prostate cancer through its ability to activate AR transcriptional function and phosphorylation of AR protein. We now demonstrate that Ack1 phosphorylates AR at Tyr-267 and -363 residues. AR is phosphorylated by heregulin-dependent HER-2 activation and Ack1 may be a downstream mediator of HER-2 in AR phosphorylation. The clinical relevance of this pathway is apparent from our finding that phosphorylated AR and activated Ack1 are found together in primary androgen-independent prostate tumor tissue specimens. The main objective of this proposal is to characterize the mechanisms by which HER-2 and Ack1 tyrosine kinases regulate AR function. Our central working hypothesis is that HER-2 and Ack1 signaling regulates the tyrosine phosphorylation status of the AR protein, leading to alterations in the assembly of the AR transcriptional complex and modulation of AR-dependent transcription. To test our hypothesis, we will concentrate on following specific aims. In Aim 1, we will characterize the effect of HER-2 signaling on assembly of the AR transcription complex. In Aim 2, we will characterize the mechanisms by which Ack1 enhances AR transcriptional activity. In Aim 3, we will characterize the functional significance of AR tyrosine phosphorylation sites. Preliminary studies supporting this proposal led to the initiation of a clinical protocol testing the concept that HER-2 kinase inhibition may be a useful therapeutic approach in prostate cancer. This proposal has the potential to yield fundamental information about prostate cancer biology and mechanisms of AR regulation by cytoplasmic signaling pathways and contribute to development of novel targeted therapy for prostate cancer.